U.S. patent application number 17/435345 was filed with the patent office on 2022-05-05 for methods and materials for biological immobilization in microfluidics.
This patent application is currently assigned to RAN Biotechnologies, Inc.. The applicant listed for this patent is RAN Biotechnologies, Inc. Invention is credited to Robert E. Lintner, Roger A. Nassar.
Application Number | 20220135962 17/435345 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220135962 |
Kind Code |
A1 |
Nassar; Roger A. ; et
al. |
May 5, 2022 |
METHODS AND MATERIALS FOR BIOLOGICAL IMMOBILIZATION IN
MICROFLUIDICS
Abstract
The present invention is directed to synthesizing and using
fluid-insoluble material complexes that capture biologicals and
remove them from samples in microscopic scale fluids, such as in
droplets, wells, and micro-wells. The present invention also
pertains to the option of detecting the captured biologicals, to
the option of modifying the captured biologicals, and to the option
of controllably releasing the captured biologicals.
Inventors: |
Nassar; Roger A.;
(Marblehead, MA) ; Lintner; Robert E.; (Amesbury,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RAN Biotechnologies, Inc |
Beverly |
MA |
US |
|
|
Assignee: |
RAN Biotechnologies, Inc.
Beverly
MA
|
Appl. No.: |
17/435345 |
Filed: |
April 17, 2020 |
PCT Filed: |
April 17, 2020 |
PCT NO: |
PCT/US2020/028776 |
371 Date: |
August 31, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62812885 |
Mar 1, 2019 |
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International
Class: |
C12N 11/04 20060101
C12N011/04; C12N 11/087 20060101 C12N011/087 |
Claims
1. A method for immobilizing a biological comprising: mixing a
fluid sample comprising the biological with a material complex
comprising a hydroxyl-, amino-, mercapto or epoxy-containing
material that is fluid-insoluble and at least one receptor selected
from lactose, lactose derivative, mono- or poly-saccharide,
heparin, chitosan, deoxyribonucleic acid, ribonucleic acid,
peptide, photoreceptor, or any combination thereof, wherein the
receptor is bound to the material; suspending the fluid sample in
at least one immiscible fluid; and separating the biological from
the fluid sample by adsorbing the biological to the material
complex.
2. The method of claim 1, wherein the biological is selected from
the group consisting of cell, cell product, tissue, tissue product,
blood, blood product, body fluid, product of body fluid, protein,
nucleic acid, vaccine, antigen, antitoxin, biological medicine,
biological treatment, virus, virus product, microorganism,
microorganism product, fungus, yeast, alga, bacterium, prokaryote,
eukaryote, Staphylococcus aureus, Streptococcus, Escherichia coli
(E. coli), Pseudomonas aeruginosa, mycobacterium, adenovirus,
rhinovirus, smallpox virus, influenza virus, herpes virus, human
immunodeficiency virus (HIV), rabies, chikungunya, severe acute
respiratory syndrome (SARS), polio, malaria, dengue fever,
tuberculosis, meningitis, typhoid fever, yellow fever, ebola,
shingella, listeria, yersinia, West Nile virus, protozoa, fungi
Salmonella enterica, Candida albicans, Trichophyton mentagrophytes,
poliovirus, Enterobacter aerogenes, Salmonella typhi, Klebsiella
pneumonia, Aspergillus brasiliensis, methicillin resistant
Staphylococcus aureus (MRSA), any derivative thereof, or any
combination thereof.
3. The method of claim 1, wherein the material is selected from the
group consisting of agarose, sand, textiles, metallic particles
(including nanoparticles), magnetic particles (including
nanoparticles), glass, fiberglass, silica, wood, fiber, plastic,
rubber, ceramic, percelain, stone, marble, cement, biological
polymers, natural polymers, synthetic polymers, poly acrylamide
polymers, poly lactic polymers, gel, colloidal gel, hydrogel, any
derivative thereof, or any combination thereof.
4. The method of claim 1, wherein the receptor is bound directly to
the material.
5. The method of claim 1, wherein the receptor is bound indirectly
to the material, via a linker.
6. The method of claim 5, wherein the linker is selected from the
group consisting of linear poly(ethylene glycol) (PEG), branched
PEG, linear poly(ethylenimine) (PEI, various ratios of
primary:secondary:tertiary amine groups), branched PEI, a dendron,
a dendrimer, a hyperbranched bis-MPA polyester-16-hydroxyl,
chitosan, any derivative thereof, or any combination thereof.
7. The method of claim 5, wherein the inter-bonding between any
combination of receptor, material, and the linker is achieved using
at least one chemical coupling reagent.
8. The method of claim 7, wherein the coupling reagent is selected
from the group consisting of 1,1'-carbonyldiimidazole (CDI),
N,N-Dicyclohexylcarbodiimide (DCC),
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC
or EDCI), or any combination thereof.
9. The method of claim 5, wherein the inter-bonding between any
combination of receptor, material and the linker is achieved using
physical attachment, or a combination of chemical and physical
attachments.
10. The method of claim 9, wherein the physical attachment is
achieved by deposition of the receptor, the linker, or a
combination thereof, onto the material in a controlled fashion, a
non-controlled fashion, or a combination thereof.
11. The method of claim 1, wherein the material is chemically
functional and the chemical functionality is amino, ammonium,
hydroxyl, mercapto, sulfone, sulfinic acid, sulfonic acid,
thiocyanate, thione, thial, thiol, carboxyl, halocarboxy, halo,
imido, anhydrido, alkenyl, alkynyl, phenyl, benzyl, carbonyl,
formyl, haloformyl, carbonato, ester, alkoxy, phenoxy, hydroperoxy,
peroxy, ether, glycidyl, epoxy, hemiacetal, hemiketal, acetal,
ketal, orthoester, orthocarbonate ester, amido, imino, imido,
azido, azo, cyano, nitrato, nitrilo, nitrito, nitro, nitroso,
pyridinyl, phosphinyl, phosphonic acid, phosphate, phosphoester,
phosphodiester, boronic acid, boronic ester, borinic acid, borinic
ester, any derivative thereof, or any combination thereof.
12. The method of claim 11, wherein the epoxy-containing material
is Poly(glycidyl methacrylate) (PGMA).
13. The method of claim 11, wherein the amino-containing material
is PGMA-NH.sub.2.
14. The method of claim 11, wherein the hydroxyl, mercapto, or
amino group is formed on a surface of the material by modifying the
substrate by a chemical transformation.
15. The method of claim 14, wherein the chemical transformation
comprising a hydrolysis reaction with an acid, a base, or a
combination thereof.
16. The method of claim 1, wherein the material complex is formed
within the fluid sample, and wherein the biological is encapsulated
or immoblized in or on the material complex.
17. The method of claim 1, further comprising separating the
immobilized biological from the fluid sample by filtration,
decantation, applying gravity or magnetic forces, flow cytometry,
fluorescence-activated cell sorter, or any combination thereof.
18. The method of claim 1, further comprising releasing the
immobilized biological from the material complexe.
19. The method of claim 18, wherein the immobilized biological is
released from the material complex by light-inducing variations,
enzymatic activity, physical variations, chemical variations, or
any combination thereof.
20-27. (canceled)
28. A material complex comprising: a hydroxyl-, amino-, mercapto or
epoxy-containing material and at least one receptor bound to the
material and selected from lactose, lactose derivative, mono- or
poly-saccharide, heparin, chitosan, deoxyribonucleic acid,
ribonucleic acid, peptide, photoreceptor, or any combination
thereof, wherein the material complex is dispersed in a first
fluid, and wherein the first fluid is suspended in an immiscible
second fluid.
Description
PRIOR APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Application No. 62/812,885, filed on Mar. 1, 2019, the
entire teachings of which are incorporated herein by reference.
FIELD
[0002] The teachings herein relate to methods and materials for
purification of biologicals, and more particularly to methods and
materials for capturing and immobilizing biologicals on
fluid-insoluble material complexes in microfluidic setups.
BACKGROUND
[0003] The use of adsorption chromatography, which includes
affinity ligand-matrix conjugates, for purification of biologicals
is well established. For example, the use of phenyl-based
adsorption chromatography for protein purification, including
purification of the in-demand monoclonal antibodies, has been
disclosed in the patent literature. Other known adsorption
chromatography processes are applied to purification of viruses
such as influenza A. However, these processes are generally not
designed for microfluidic setups.
[0004] Accordingly, there remains a critical need for purification
of biological materials in microfluidic setups.
SUMMARY
[0005] The present invention is directed to methods and materials
for immobilizing biologicals using fluid-insoluble material
complexes that specifically capture microorganisms, microorganism
products, proteins, nucleic acids, peptides, and other biologicals
within small volumes of fluids on the order of micro-, nano-,
pico-liter, or even smaller. It also pertains to the option of
controllably releasing the captured biologicals under certain
conditions.
[0006] In one aspect, a method of immobilizing biologicals is
discussed, which includes mixing a sample containing biologicals
with material complexes, followed by generating an emulsion of
small-volume droplet or droplets which contain the complexed
biologicals, and which are suspended in a continuous phase that is
immiscible with the phase of the droplets. The material complexes
can include hydroxyl-, amino-, mercapto- or epoxy-containing
materials that are fluid-insoluble and at least one receptor bound
to the materials. The biologicals can include for example any of a
cell, tissue, tissue product, blood, blood product, protein,
nucleic acids, vaccine, antigen, antitoxin, virus, microorganism,
fungus, yeast, alga, and bacterium. If desired, the immobilized
biologicals can then be extracted from the material complex, such
as by elution. In the case of the virus, the extracted biological
can then be included in a vaccine treatment. In the case of the
protein, the extracted biological can then be included in a vaccine
or therapeutic treatment.
[0007] In another aspect, a method of immobilizing biologicals is
disclosed, which includes generating two separate emulsions
followed by mixing them: the first emulsion is made from
small-volume droplet or droplets which contain the biologicals, and
which are suspended in a continuous phase that is immiscible with
the phase of the droplets; and the second emulsion is made from
small-volume droplet or droplets which contain the material
complexes and which are suspended in a continuous phase that is
immiscible with the phase of the droplets. The two emulsions are
then mixed allowing the controlled or un-controlled fusion of two
or more droplets from these two emulsions, where at least one
droplet from each emulsion is represented. The new fused droplets
can simultaneously contain biologicals and material complexes,
allowing for immobilization of the biologicals on the material
complexes. The material complexes can include hydroxyl-, amino-,
mercapto- or epoxy-containing materials that are fluid-insoluble
and at least one receptor bound to the materials. The biologicals
can include, for example, any of a cell, tissue, tissue product,
blood, blood product, protein, nucleic acids, vaccine, antigen,
antitoxin, virus, microorganism, fungus, yeast, alga, and
bacterium. If desired, the immobilized biologicals can then be
extracted from the material complex, such as by elution. In the
case of the virus, the extracted biological can then be included in
a vaccine treatment. In the case of the protein, the extracted
biological can then be included in a vaccine or therapeutic
treatment.
[0008] In yet another aspect, a method according to the present
teachings can include mixing the starting materials of material
complexes with biologicals, followed by generating an emulsion of
small-volume droplet or droplets which contain the starting
materials and the biologicals, and which are suspended in a
continuous phase that is immiscible with the phase of the droplets.
The next step is allowing the in-situ formation of material
complexes, while simultaneously immobilizing biologicals on the
material complexes. The material complexes can include hydroxyl-,
amino-, mercapto- or epoxy-containing materials, hydrogels,
poly-lactic-containing polymers, that are fluid-insoluble and at
least one receptor bound to the materials. The biologicals can
include for example any of a cell, tissue, tissue product, blood,
blood product, protein, nucleic acids, vaccine, antigen, antitoxin,
virus, microorganism, fungus, yeast, alga, and bacterium. If
desired, the immobilized biologicals can then be extracted from the
material complex, such as by elution. In the case of the virus, the
extracted biological can then be included in a vaccine treatment.
In the case of the protein, the extracted biological can then be
included in a vaccine or therapeutic treatment.
[0009] In yet another aspect, methods and materials for forming the
aforementioned materials, material complexes, mixtures,
compositions, composites, emulsions, or any combination thereof,
and for purifying, immobilizing, capturing, and separating the
aforementioned biologicals in wells and micro-wells instead of
droplets are disclosed.
[0010] In another aspect, a method for immobilizing a biological is
disclosed, which includes mixing a fluid sample comprising the
biological with a material complex comprising a hydroxyl-, amino-,
mercapto or epoxy-containing material that is fluid-insoluble and
at least one receptor selected from lactose, lactose derivative,
mono- or poly-saccharide, heparin, chitosan, deoxyribonucleic acid,
ribonucleic acid, peptide, photoreceptor, or any combination
thereof. The receptor can be bound to the material. The method can
also include suspending the fluid sample in at least one immiscible
fluid and separating the biological from the fluid sample by
adsorbing the biological to the material complex.
[0011] In some aspects, the biological can be selected from the
group consisting of cell, cell product, tissue, tissue product,
blood, blood product, body fluid, product of body fluid, protein,
nucleic acid, vaccine, antigen, antitoxin, biological medicine,
biological treatment, virus, virus product, microorganism,
microorganism product, fungus, yeast, alga, bacterium, prokaryote,
eukaryote, Staphylococcus aureus, Streptococcus, Escherichia coli
(E. coli), Pseudomonas aeruginosa, mycobacterium, adenovirus,
rhinovirus, smallpox virus, influenza virus, herpes virus, human
immunodeficiency virus (HIV), rabies, chikungunya, severe acute
respiratory syndrome (SARS), polio, malaria, dengue fever,
tuberculosis, meningitis, typhoid fever, yellow fever, ebola,
shingella, listeria, yersinia, West Nile virus, protozoa, fungi
Salmonella enterica, Candida albicans, Trichophyton mentagrophytes,
poliovirus, Enterobacter aerogenes, Salmonella typhi, Klebsiella
pneumonia, Aspergillus brasiliensis, methicillin resistant
Staphylococcus aureus (MRSA), any derivative thereof, or any
combination thereof.
[0012] In some aspects, the material can be selected from the group
consisting of agarose, sand, textiles, metallic particles
(including nanoparticles), magnetic particles (including
nanoparticles), glass, fiberglass, silica, wood, fiber, plastic,
rubber, ceramic, percelain, stone, marble, cement, biological
polymers, natural polymers, synthetic polymers, poly acrylamide
polymers, poly lactic polymers, gel, colloidal gel, hydrogel, any
derivative thereof, or any combination thereof.
[0013] In some aspects, the receptor can be bound directly to the
material. In other aspects, the receptor can be bound indirectly to
the material, e.g., via a linker.
[0014] In some aspects, the linker can be selected from the group
consisting of linear poly(ethylene glycol) (PEG), branched PEG,
linear poly(ethylenimine) (PEI, various ratios of
primary:secondary:tertiary amine groups), branched PEI, a dendron,
a dendrimer, a hyperbranched bis-MPA polyester-16-hydroxyl,
chitosan, any derivative thereof, or any combination thereof.
[0015] In some aspects, the inter-bonding between any combination
of receptor, material, and the linker can be achieved using at
least one chemical coupling reagent. In these aspects and in other
aspects, the coupling reagent can be selected from the group
consisting of 1,1'-carbonyldiimidazole (CDI),
N,N'-Dicyclohexylcarbodiimide (DCC),
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC
or EDCI), or any combination thereof.
[0016] In some aspects, the inter-bonding between any combination
of receptor, material, and the linker can be achieved using
physical attachment, chemical attachment, or a combination of
chemical and physical attachments. In these aspects and other
aspects, the physical attachment can be achieved by deposition of
the receptor, the linker, or a combination thereof, onto the
material in a controlled fashion, a non-controlled fashion, or a
combination thereof.
[0017] In some aspects, the material can be chemically functional
and the chemical functionality can be amino, ammonium, hydroxyl,
mercapto, sulfone, sulfinic acid, sulfonic acid, thiocyanate,
thione, thial, thiol, carboxyl, halocarboxy, halo, imido,
anhydrido, alkenyl, alkynyl, phenyl, benzyl, carbonyl, formyl,
haloformyl, carbonato, ester, alkoxy, phenoxy, hydroperoxy, peroxy,
ether, glycidyl, epoxy, hemiacetal, hemiketal, acetal, ketal,
orthoester, orthocarbonate ester, amido, imino, imido, azido, azo,
cyano, nitrato, nitrilo, nitrito, nitro, nitroso, pyridinyl,
phosphinyl, phosphonic acid, phosphate, phosphoester,
phosphodiester, boronic acid, boronic ester, borinic acid, borinic
ester, any derivative thereof, or any combination thereof. In these
aspects and other aspects, the epoxy-containing material can be
Poly(glycidyl methacrylate) (PGMA) and the amino-containing
material can be PGMA-NH.sub.2.
[0018] In some aspects, the hydroxyl, mercapto, or amino group can
be formed on a surface of the material by modifying the substrate
by a chemical transformation. In these aspects, the chemical
transformation can comprise a hydrolysis reaction with an acid, a
base, or a combination thereof.
[0019] In some aspects, the material complex can be formed within
the fluid sample and the biological can be encapsulated or
immoblized in or on the material complex.
[0020] In some aspects, a method for immobilizing a biological is
disclosed, which includes separating an immobilized biological from
a fluid sample by filtration, decantation, applying gravity or
magnetic forces, flow cytometry, fluorescence-activated cell
sorter, or any combination thereof. In these aspects and other
aspects, the method can include releasing the immobilized
biological from the material complex by, for example,
light-inducing variations, enzymatic activity, physical variations,
chemical variations, or any combination thereof. In these aspects
and other aspects, the method can include releasing the immobilized
biological from the material complexby, for example, temperature
variations, irradiation variations, mechanical variations,
thermodynamic variations, thermomechanic variations, or any
combination thereof. In these aspects and other aspects, the method
can include releasing the immobilized biological from the material
complexby, for example, variations in pH values, concentration of
chemicals, concentration of ions, concentration of sodium chloride,
or any combination thereof.
[0021] In some aspects, a method for immobilizing a biological is
disclosed, wherein the method can be part of a process, production,
operation, kit, or application of medicine, vaccine, medical
device, diagnostic equipment and techniques, implant, glove, mask,
textile, surgical drape, tubing, surgical instrument, safety gear,
fabric, apparel item, floor, handle, wall, sink, shower, tub,
toilet, furniture, wall switch, toy, athletic equipment, playground
equipment, shopping cart, countertop, appliance, railing, door, air
filter, air processing equipment, water filter, water processing
equipment, pipe, phone, cell phone, remote control, computer,
mouse, keyboard, touch screen, leather, cosmetic, cosmetic making
equipment, cosmetic storage equipment, personal care item, personal
care item making equipment, personal care storage equipment, animal
care item, animal care item making equipment, animal care storage
equipment, veterinary equipment, powder, cream, gel, salve, eye
care item, eye care item making equipment, eye care storage
equipment, contact lens, contact lens case, glasses, jewelry,
jewelry making equipment, jewelry storage equipment, utensil, dish,
cup, container, object display container, food display container,
food package, food processing equipment, food handling equipment,
food transportation equipment, food storage equipment, food vending
equipment, animal housing, farming equipment, animal food handling
equipment, animal food storage space, animal food processing
equipment, animal food storage equipment, animal food container,
air vehicle, land vehicle, water vehicle, water storage space,
water storage equipment, water storage container, water processing
equipment, water storage equipment, water filter, air filter, or
any combination thereof.
[0022] In another aspect, a method for protecting an object against
microbial infection, microbial colonization, or microbial
trans-infection is disclosed, which includes providing to the
object a microbial barrier according to one or more of the methods
disclosed herein.
[0023] In some aspects, a method for immobilizing a biological is
disclosed, which include detecting the immobilized biological,
modifying the immobilized biological, or detecting and modifying
the immobilized biological. In these aspects and other aspects, the
modified immobilized biological can be released from the material
complex according to one or more methods disclosed herein.
[0024] In some aspects, the immiscible fluid can be in a well.
[0025] In another aspect, a material complex is disclosed, which
includes a hydroxyl-, amino-, mercapto or epoxy-containing material
and at least one receptor bound to the material and selected from
lactose, lactose derivative, mono- or poly-saccharide, heparin,
chitosan, deoxyribonucleic acid, ribonucleic acid, peptide,
photoreceptor, or any combination thereof. In this aspect and other
aspects, the material complex can be dispersed in a second fluid.
In this aspect and other aspects, the first fluid can be suspended
in an immiscible second fluid.
[0026] These and other aspects of the applicants' teaching are set
forth herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The foregoing and other objects and advantages of the
invention will be appreciated more fully from the following further
description, with reference to the accompanying drawings. The
skilled person in the art will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the applicants' teachings in
any way.
[0028] FIG. 1 schematically illustrates three different embodiments
of fluid-insoluble material cores that are complexed with receptors
either directly or indirectly through linkers in accordance with
various aspects of the applicants' teachings;
[0029] FIG. 2 schematically illustrates examples of direct
attachment of receptors to materials in accordance with various
aspects of the applicants' teachings;
[0030] FIGS. 3A, 3B, and 3C schematically illustrate examples of
attachment of receptors to materials via linkers in accordance with
various aspects of the applicants' teachings;
[0031] FIG. 4 schematically illustrates a general route for
covalent coupling when using 1,1'-carbonyldiimidazole in accordance
with various aspects of the applicants' teachings;
[0032] FIG. 5 schematically illustrates the emulsification of
biologicals immobilized on fluid-insoluble material complexes in
accordance with various aspects of the applicants' teachings;
[0033] FIG. 6 schematically illustrates the fusion of two
emulsions, Emulsion A made from droplets containing fluid-insoluble
material complexes and Emulsion B made from droplets containing
biologicals, in accordance with various aspects of the applicants'
teachings;
[0034] FIG. 7 schematically illustrates engineered emulsification
of homogeneously-sized droplets, each containing biologicals
immobilized on fluid-insoluble material complexes, using a
microfluidic chip in accordance with various aspects of the
applicants' teachings;
[0035] FIG. 8 schematically illustrates the fusion of two
engineered emulsions of homogeneously-sized droplets, the first set
of droplets contains fluid-insoluble material complexes and the
second set of droplets contains biologicals, in accordance with
various aspects of the applicants' teachings;
[0036] FIG. 9 is a diagram of the chemical derivatization of
materials monitored by recombinant HA binding assays in accordance
with various aspects of the applicants' teachings;
[0037] FIG. 10 is a diagram of the concentration of the captured
virus in accordance with various aspects of the applicants'
teachings; and
[0038] FIG. 11 is a diagram of the adsorbed virus and initial virus
in accordance with various aspects of the applicants'
teachings.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention is directed to the methods and
materials for capturing and immobilizing biologicals on
fluid-insoluble material complexes in microfluidic setups. It also
pertains to the option of controllably releasing the captured
biologicals under specific conditions.
[0040] Certain exemplary embodiments will now be described to
provide an overall understanding of the principles of the
structure, function, manufacture, and use of the methods and
materials disclosed herein. One or more examples of these
embodiments are illustrated in the accompanying drawings. Those
skilled in the art will understand that the devices and methods
specifically described herein and illustrated in the accompanying
drawings are non-limiting exemplary embodiments and that the scope
of the present invention is defined solely by the claims. The
features illustrated or described in connection with one exemplary
embodiment may be combined with the features of other embodiments.
Such modifications and variations are intended to be included
within the scope of the present invention.
[0041] All publications, patents, and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety. As used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural references unless the content clearly dictates otherwise.
The terms used in this invention adhere to standard definitions
generally accepted by those having ordinary skill in the art. In
case any further explanation might be needed, some terms have been
further elucidated below.
[0042] The term "biologicals" as used herein refers to living
organisms and their products, including, but not limited to, cell,
tissue, tissue product, blood, blood product, protein,
deoxyribonucleic acid, ribonucleic acid, nucleic acid, vaccine,
antigen, antitoxin, viruses, microorganism, fungi, yeast, algae,
bacteria, derivative thereof, or any combination thereof. One
example of biological can include microorganism, such as pathogenic
or non-pathogenic bacteria. Other examples of biologicals can
include viruses, viral products, virus-imitating entities,
derivative thereof, or any combination thereof.
[0043] The term "about" as used herein denotes a variation of at
most 10% around a numerical value.
[0044] In one embodiment, fluid-insoluble materials can be
complexed with microoganism-capturing groups (also called
"receptors"), the structures of which are drawn from natural
cellular receptors, antibodies, or simply from available data
describing microoganism interaction with soluble molecules. The
receptors can be directly attached to the material (FIG. 1, Mode A)
or through a linker (FIG. 1, Mode B). In order to protect the
integrity of the molecular structure of the subject material
complexes, particularly when re-cycling is a requirement, one
method of inter-connecting the receptors, linkers and materials can
be via covalent bonding. For certain applications where added
structural stability is not needed, for example in single use
material complexes, physical bonding can substitute covalent
bonding. The receptors play a direct role by capturing the
microorganims through physical bonding, e.g., by hydrogen bonding.
One role of linkers is to position the receptors at an active
distance from the core of the material. By distancing the receptors
from the core of the material, the receptors can easily access the
target microorganisms. Another role for the linkers, particularly
when they are branched, is to increase the density of the receptors
on the surface of the material (FIG. 1, Mode C). In many
embodiments, an increase in the density of receptors correlates
with an increase in the capacity of capturing higher concentrations
of microorganisms.
[0045] Examples of the three main components of the material
complexes are: 1) materials: agarose, sand, textiles (e.g.,
cellulose/cotton, wool, nylon, polyester), metallic particles
(e.g., nanoparticles), magnetic particles (e.g., nanoparticles),
glass, fiberglass, silica, wood, fiber, plastic, rubber, ceramic,
percelain, stone, marble, cement, biological polymers, natural
polymers and synthetic polymers (e.g., PGMA), derivative thereof,
or any combination thereof; 2) receptors: lactose (natural and
synthetic) and its derivatives (e.g., sialyllactose), mono- and
poly-saccharides (natural and synthetic), heparin and chitosan,
derivative thereof, or any combination thereof; and 3) linkers:
linear and branched polymers, such as poly(ethylene glycol) (PEG)
and poly(ethylenimine) (PEI, various ratios of
primary:secondary:tertiary amine groups), (e.g., multi-arm branched
PEG-amines), dendrons and dendrimers (e.g., hyperbranched bis-MPA
polyester-16-hydroxyl), chitosan, derivative thereof, or any
combination thereof. Each of the material complexes may incorporate
the material and the receptor components. However, incorporating
the linker component is optional.
[0046] Metal materials suitable for use in the invention include,
for example, stainless steel, nickel, titanium, tantalum, aluminum,
copper, gold, silver, platinum, zinc, Nitinol, Inconel, iridium,
tungsten, silicon, magnesium, tin, alloys, coatings containing any
of the foregoing, galvanized steel, hot dipped galvanized steel,
electrogalvanized steel, annealed hot dipped galvanized steel,
derivative thereof, or any combination thereof.
[0047] Glass materials suitable for use in the invention include,
for example, soda lime glass, strontium glass, borosilicate glass,
barium glass, glass-ceramics containing lanthanum, derivative
thereof, or any combination thereof.
[0048] Sand materials suitable for use in the invention include,
for example, sand comprising silica (e.g., quartz, fused quartz,
crystalline silica, fumed silica, silica gel, and silica aerogel),
calcium carbonate (e.g., aragonite), derivative thereof, or any
combination thereof. The sand can comprise other components, such
as minerals (e.g., magnetite, chlorite, glauconite, gypsum,
olivine, garnet), metal (e.g., iron), shells, coral, limestone,
rock, derivative thereof, or any combination thereof.
[0049] Wood materials suitable for the invention include, for
example, hard wood, soft wood, and materials engineered from wood,
wood chips, and fiber (e.g., plywood, oriented strand board,
laminated veneer lumber, composites, strand lumber, chipboard,
hardboard, and medium density fiberboard), derivative thereof, or
any combination thereof. Types of wood include alder, birch, elm,
maple, willow, walnut, cherry, oak, hickory, poplar, pine, fir, or
any combination thereof.
[0050] Fiber materials suitable for use in the invention include,
for example, natural fibers (e.g., derived from an animal,
vegetable, or mineral) and synthetic fibers (e.g., derived from
cellulose, mineral, or polymer). Suitable natural fibers include,
for example, cotton, hemp, jute, flax, ramie, sisal, bagasse, wood
fiber, silkworm silk, spider silk, sinew, catgut, wool, sea silk,
wool, mohair, angora, and asbestos. Suitable synthetic fibers
include, for example, rayon, modal, Lyocell, metal fiber (e.g.,
copper, gold, silver, nickel, aluminum, iron), carbon fiber,
silicon carbide fiber, bamboo fiber, seacell, nylon, polyester,
polyvinyl chloride fiber (e.g., vinyon), polyolefin fiber (e.g.,
polyethylene, polypropylene), acrylic polyester fiber, aramid,
spandex, or any combination thereof.
[0051] Natural polymer materials suitable for use in the invention
include, for example, a polysaccharide (e.g., cotton, cellulose),
shellac, amber, wool, silk, natural rubber, and a biopolymer (e.g.,
a protein, an extracellular matrix component, collagen), or any
combination thereof.
[0052] Synthetic polymer materials suitable for use in the
invention include, for example, polyvinylpyrrolidone, acrylics,
acrylonitrile-butadiene-styrene, poly acrylonitrile, acetals,
polyphenylene oxides, polyimides, polystyrene, polypropylene,
polyethylene, polytetrafluoroethylene, polyvinylidene fluoride,
polyvinyl chloride, polyethylenimine, polyesters, polyethers,
polyamide, polyorthoester, polyanhydride, polysulfone, polyether
sulfone, polycaprolactone, polyhydroxy-butyrate valerate,
polylactones, polyurethanes, polycarbonates, polyethylene
terephthalate, copolymers, derivative thereof, or any combination
thereof.
[0053] Typical rubber materials suitable for use in the invention
include, for example, silicones, fluorosilicones, nitrile rubbers,
silicone rubbers, polyisoprenes, sulfur-cured rubbers,
butadiene-acrylonitrile rubbers, isoprene-acrylonitrile rubbers,
derivative thereof, or any combination thereof.
[0054] Ceramic materials suitable for use in the invention include,
for example, boron nitrides, silicon nitrides, aluminas, silicas,
the like, derivative thereof, or any combination thereof.
[0055] Stone materials suitable for use in the invention include,
for example, granite, quartz, quartzite, limestone, dolostone,
sandstone, marble, soapstone, serpentine, derivative thereof, and
any combination thereof.
[0056] Exemplary receptors can include: 1) heparin, a negatively
charged polymer that can mimic innate glycosaminoglycanes found in
the memebranes of host cells. It is commercially available as
heparin sodium which is extracted from porcine intestinal mucosa
and is approved as blood anti-coagulant. Also, non-animal-derived
synthetic heparin-mimicking sulfonic acid polymers can act in a
similar fashion to natural heparin; 2) chitosan, an ecologically
friendly bio-pesticide that can ligate to a variety of
microorganisms and proteins. It is also used as a hemostatic agent
and in transdermal drug delivery; and 3) lactose, a by-product of
the dairy industry. It is widely available and produced annually in
millions of tons. Lactose can also be synthesized by
condensation/dehydration of the two sugars, galactose and glucose,
including all their isomers. Exemplary receptors can also include
heparin derivative, chitosan derivative, lactose derivative, or any
combination thereof.
[0057] Exemplary materials can include: 1) sand, an affordable and
widely available material. In addition, complexed sand could easily
replace non-complexed sand in established technologies such as
drinking water purification; 2) agarose, particularly
Sepharose.RTM., a beaded polysaccharide polymer extracted from
seaweed. They are also widely available and used in chromatography
to separate biomolecules; and 3) PGMA, a synthetic polymer produced
from Glycidyl methacrylate, which is an ester of methacrylic acid
and a common monomer used in the production of epoxy materials.
[0058] Exemplary linkers can include: 1) chitosan (see its
description as a receptor); 2) poly(ethylene glycol)(PEG) and its
derivatives, produced from ethylene oxides with many different
chemical, biological, commercial, and industrial uses; and 3)
dendrons and dendrimers, relatively new molecules. They are
repetitively branched molecules using a small number of starting
reagents. They are commonly used in drug delivery and sensors. Some
suitable examples of dendrons and dendrimers include, without
limitation, hydroxyl-terminated polyester dendrons,
amine-terminated carbosilane dendrons, and hydroxyl-terminated
polyether dendrons.
[0059] In one aspect, the receptors can be directly attached to the
material (FIG. 2) or through linkers (FIG. 3) via chemical
coupling. One type of coupling reagent is 1,1'-carbonyldiimidazole
(CDI). The coupling reagent may also be
N,N'-Dicyclohexylcarbodiinide (DCC) or
N-(3-Dimethylaininopropyl)-N'-ethylcarbodiimide hydrochloride (EDC
or EDCT).
[0060] An exemplary coupling reagent is CDI. Basic protonated end
groups, such as hydroxyl groups (R--OH) in sand and Sepharose.RTM.
and tertiary amine groups (R--NH.sub.2) in PGMA-diaminobutane,
readily react with CDI to form an ester or amide link. The
resulting imidazole-substituted derivatives are reacted with
hydroxyl-terminated receptors yielding either carbonates
[R--O--C(O)--O-receptor] or carbamates [R--N(H)--C(O)--O-receptor].
The resulting imidazole-substituted derivatives can also be reacted
with amine-terminated receptors yielding urea derivatives
[R--N(H)--C(O)--N(H)-receptor] (FIG. 4). Due to the formation of a
covalent bound between the receptor and the material (via direct
bonding or through a linker), the structure of the bound receptor
is different compared to the structure of the commercially
available free receptor. For example, as depicted in FIG. 6, the
receptor can lose a hydrogen atom upon reaction with the
immidazole-substituted derivatives to form a receptor-carbonate,
receptor-carbamate, or receptor-urea derivative.
[0061] When an appropriate functional group is not present on the
surface of the material, a suitable functional group can be made
available to the surface by a chemical transformation. In general,
a chemical transformation can be hydrolysis, oxidation (e.g., using
Collins reagent, Dess-Martin periodinane, Jones reagent, and
potassium permanganate), reduction (e.g., using sodium borohydride
or lithium aluminum hydride), alkylation, deprotonation,
electrophilic addition (e.g., halogenation, hydrohalogenation, and
hydration), hydrogenation, esterification, elimination reaction
(e.g., dehydration), nucleophilic substitution, radical
substitution, or a rearrangement reaction. If needed, more than one
chemical transformation, successively or simultaneously, can be
used to provide a suitable functional group or a heterogeneous
group of functional groups of various identities. Alternatively, a
monomer with a desired functional group can be grafted to the
material.
[0062] In some embodiments, the chemical transformation is
hydrolysis. Generally, hydrolysis is performed with water in the
presence of a strong inorganic, organic, or organo-metallic acid
(e.g., strong inorganic acid, such as hydrochloric acid, sulfuric
acid, phosphoric acid, nitric acid, hydroiodic acid, hydrobromic
acid, chloric acid, and perchloric acid) or strong inorganic,
organic, or organo-metallic base (e.g., Group I and Group II
hydroxides, such as lithium hydroxide, sodium hydroxide, potassium
hydroxide, rubidium hydroxide, cesium hydroxide, magnesium
hydroxide, calcium hydroxide, and barium hydroxide; ammonium
hydroxide; and sodium carbonate). For example, a material
comprising an acyl halide can undergo hydrolysis to form a
carboxylic acid.
[0063] In some embodiments, the chemical transformation is a
substitution reaction where one functional group is replaced with
another. For example, a material comprising a haloalkyl group can
react with a strong base to form a hydroxy group.
[0064] In other aspects, the chemical transformation is alkylation,
hydrogenation, or reduction. For example, a material comprising a
hydroxy or haloalkyl (e.g., iodoalkyl or bromoalkyl) moiety can be
reacted with ammonia to form an amino group. A material comprising
a haloalkyl moiety also can be converted to a mercapto group by
S-alkylation using thiourea. A material comprising a nitrile can be
hydrogenated to form an amino group. A material comprising an amido
group can be reduced (e.g., in the presence of lithium aluminum
hydride) to form an amino group. A material comprising a formyl or
keto group can be reduced to form an amino or hydroxy group.
Multiple homogeneous or heterogeneous transformations can be
applied simultaneously or successively.
[0065] A variety of material complexes can be used in the present
invention such as the ones disclosed in U.S. Pat. No. 10,105,681
and US Pub. No. 2016/0010136 which are herein incorporated by
reference in their entirety. Material complexes comprise, for
example, lactose-Sepharose, lactose-sand, lactose-PGMA,
heparin-Sepharose, heparin-sand, heparin-PGMA,
lactose-[branching]-Sepharose, lactose-[branching]-sand,
lactose-[branching]-PGMA, heparin-[branching]-Sepharose,
heparin-[branching]-sand, heparin-[branching]-PGMA, and derivatives
thereof. The material complexes can be formed by any suitable
method using suitable temperatures (e.g., room temperature and
reflux), reaction times, solvents, catalysts, and concentrations.
In some aspects, an excess amount of linkers and receptors can be
used to ensure an effective amount of receptors in the material
complexes.
[0066] In another aspect, attachments amongst receptors, linkers,
and materials can be secured physically. This is achieved by mixing
receptors or linkers, or any combination thereof, dissolved in one
or more solvents with the materials, then allowing the one or more
solvents to evaporate in air, under vacuum, or a combination
thereof.
[0067] The receptors may also reversibly interact with the target
biologicals, such as micro-organisms or viruses. The biologicals
can be desorbed from the receptors, such as through elution.
Eluents such as higher-than-physiological sodium chloride solutions
and lactose-containing solutions are capable of desorbing the
biologicals from the material complexes.
[0068] Depicted in FIGS. 2 and 3, one exemplary receptor is
lactose. Immobilized lactose can be used for capturing a high titer
of influenza A virus. Furthermore, lactose-PGMA combination is also
an exemplary material.
[0069] The material complexes can be used for the capture of
biologicals in fluids. These material complexes should not dissolve
in the aforementioned fluids.
[0070] The disclosed methods and material complexes may be used in
a number of applications including, for example: 1)
pharmaceuticals: culturing microorganisms, inoculating
microorganisms, purification of vaccines, proteins, including
monoclonal antibodies (MAbs), and other biologicals; 2)
diagnostics: increasing the concentration of target biologicals in
samples leading to increase in sensitivity in existing and novel
diagnostic tools, or including materials that change color upon
binding a biological molecule or exhibit a signal indicating their
binding to biologicals and allowing simple point-of-use
diagnostics; 3) prophylactics: trapping biologicals prior to
infection or contamination (e.g. face masks, air purifiers, and
gloves); 4) therapeutics: disinfection of blood and its products,
extracorporeal dialysis, disinfection of intestinal fluids, and
controlling the biological composition of life-sustaining fluids;
and 5) environmental: removing biologicals from water and other
fluids in the environment, including air.
[0071] In one embodiment, the disclosed methods and material
complexes can be used for vaccine purification. Current vaccine
purification techniques use a combination of membrane separation
(e.g., ultrafiltration) and chromatographic separation (e.g., size
exclusion and ion exchange). While the overall purity is above
about 90%, the yield is only about 50%. The disclosed methods and
material complexes can substitute the separations based on size
exclusion, ion exchange chromatography, or a combination thereof.
When the disclosed methods and material complexes show high
selectivity towards target biologicals, it is possible that the
disclosed methods and material complexes could substitute
chromatograpic separations, membrane separation, other filtration
steps, or any combination thereof.
[0072] In another embodiment, the disclosed methods and material
complexes can be used in microfluidic setups. Such setups have the
advantage of allowing the execution and study of reactions and
interactions on very small microscopic scale, which leads to
amplified signals and minimized noises due to irrelevant reactions
and interactions.
[0073] In another embodiment, the disclosed methods and material
complexes combined with target biologicals can be combined with a
non-miscible fluid (FIG. 5). The mixture can then be emulsified via
shaking, vortexing, other technical emulsification procedures, or
any combination thereof. The resulting emulsion can be composed of
droplets suspended in the non-miscible fluid. Each droplet can
contain material complexes, target biologicals, or a combination
thereof.
[0074] Non-miscible fluids suitable for use in the invention
include, for example, mineral oils, hydrocarbon oils, vegetable
oils, parafin oils, fluorinated oils, fully fluorinated oils,
partially fluorinated oils, any derivative thereof, or any
combination thereof.
[0075] In another embodiment, the disclosed methods and material
complexes can be combined with a non-miscible fluid to form
Emulsion A; and the disclosed methods and biologicals can be
combined with a non-miscible fluid to form Emulsion B (FIG. 6). The
emulsifications can be achieved via shaking, vortexing, other
technical emulsification procedures, or any combination thereof.
The two resulting emulsions, A and B, can be combined and droplets
can be controllably or un-controllably merged, facilitating
potential interactions between material complexes and
biologicals.
[0076] In another embodiment, the disclosed methods and material
complexes combined with target biologicals can be combined with a
non-miscible fluid in a controlled or engineered method to form an
engineered emulsion (FIG. 7). An example of controlled or
engineered method is by using a microfluidic chip. The resulting
emulsion is a mixture of droplets containing material complexes,
target biologicals, or a combination thereof.
[0077] In another embodiment, the disclosed methods and material
complexes can be combined with a non-miscible fluid in a controlled
or engineered method to form droplets containing the material
complexes; and the disclosed methods, materials, and/or biologicals
can be combined with a non-miscible fluid in a controlled or
engineered method to form droplets containing biologicals (FIG. 8).
The resulting droplets can be controllably or un-controllably
merged, so each droplet can contain material precursors, material
complexes, target biologicals, or any combination thereof.
EXAMPLES
[0078] The following Examples further illustrate the salient
aspects of the invention. The Examples are provided only for
illustration purposes and are not intended to necessarily indicate
the optimal ways of practicing the invention or optimal results
that can be obtained.
Example 1
[0079] As an example of the experimental work, the synthesis of
lactose-sand (FIG. 2-A) followed these steps: 5 grams of fine sand
was rinsed with 20 ml DI water while on a medium frit filter. They
were then mixed with 10 ml pH 8.5 (20 mM) borate buffer and allowed
to stir for 10 minutes at room temperature. Thirty nine mg of
1,1'-carbonyldiimidazole (0.24 mmol, MW 162.15) was then added to
the suspension and allowed to react for 2 hours before adding 190
mg of .beta.-D-lactose (0.55 mmol). The resulting mixture was
allowed to stir for 4 days at room temperature. The final
suspension was filtered and the solid was rinsed with de-ionized
(DI) water. The wetness of the solid was preserved.
Example 2
[0080] As an example of the experimental work, the synthesis of
lactose-Sepharose.RTM. (FIG. 2-B) followed these steps: 5 grams of
wet Sepharose (ca. 5 wt. % in water) was mixed with 10 ml pH 8.5
(20 mM) borate buffer and allowed to stir for 10 minutes at room
temperature. Thirty nine mg of 1,1'-carbonyldiimidazole (0.24 mmol,
MW 162.15) was then added to the suspension and allowed to react
for 2 hours before adding 190 mg of .beta.-D-lactose (0.55 mmol).
The resulting mixture was allowed to stir for 4 days at room
temperature. The final suspension was filtered and the solid was
rinsed with 100 ml DI water. The wetness of the solid was
preserved.
Example 3
[0081] As an example of the experimental work, the synthesis of
lactose-PGMA (FIG. 2-C) followed these steps: A 100 ml single neck
round bottom flask and a magnetic bar were dried under vacuum while
hot. Fifty ml dry tetrahydrofuran was added followed by 1.24 g (14
mmol) of 1,4-diaminobutane. While stirring the solution, 200 mg
PGMA (1.4 mmol equivalents of the repeat unit) was added. The
solution was then allowed to stir at room temperature for 10 min
before starting the in-situ evacuation into a cold trap, using the
vacuum line. The reaction flask was gently heated using a heating
gun in order to ensure the removal of all volatile reagents. To the
resulting oil-like product, 50 ml DI water were added leading to
the precipitation of a white film-like solid. This solid was then
filtered on a medium frit and rinsed with 300 ml DI water. The
yield was 0.529 g of PGMA-NH.sub.2. The final polymer was
efficiently dried and stored at low temperature.
[0082] One hundred and ten mg of the resulting intermediate,
PGMA-NH.sub.2, was mixed with 10 ml pH 8.5 20 mM borate buffer and
allowed to stir for few minutes at room temperature. Nineteen mg of
1,1'-carbonyldiimidazole was then added to the suspension and
allowed to stir for 1 hour before adding 0.055 g of
.beta.-D-lactose. The final mixture was allowed to stir for two
days at room temperature followed by filtering through a medium
frit and rinsing with 50 ml DI water. The wetness of the solid was
preserved.
Example 4
[0083] As an example of the experimental work, the synthesis of
lactose-[branching]-sand (FIG. 3-A) followed these steps: Five
grams of fine sand was vigorously stirred with 20 ml DI water, then
filtered through a medium frit. They were then mixed with 10 ml pH
8.5 20 mM borate buffer and allowed to stir for few minutes at room
temperature. Sixteen mg of 1,1'-carbonyldiimidazole (0.1 mmol, MW
162.15) was then added to the suspension and allowed to stir for 2
more hours before adding 0.25 g of Hyperbranched bis-MPA
polyester-16-hydroxyl (0.1425 mmol, 2.28 mmol.eq. OH). After two
additional hours, 0.37 g (2.28 mmol) of 1,1'-carbonyldiimidazole
was added to the suspension and allowed to stir for 2 more hours
before adding 3.9 g (11.4 mmol) of .beta.-D-lactose. Five ml of the
pH 8.5 borate buffer was then added. The final "almost clear"
mixture was allowed to stir for two days at room temperature. The
final solution was filtered through a medium frit and rinsed with
50 ml DI water, isolating 4.8943 g of sand complex the color of
which was similar to that of the starting sand. The wetness of the
solid was preserved.
Example 5
[0084] As an example of the experimental work, the synthesis of
lactose-[branching]-Sepharose (FIG. 3-B) followed these steps: One
gram of wet Sepharose (ca. 5 wt. % in water) was mixed with 10 ml
pH 8.5 20 mM borate buffer and allowed to stir for few minutes at
room temperature. Thirty two mg of 1,1'-carbonyldiimidazole (0.2
mmol, MW 162.15) was then added to the suspension and allowed to
stir for 2 more hours before adding 0.5 g of Hyperbranched bis-MPA
polyester-16-hydroxyl (0.285 mmol, 4.56 mmol.eq. OH). After two
additional hours, 0.74 g (4.56 mmol) of 1,1'-carbonyldiimidazole
was added to the suspension and allowed to stir for 2 hours before
adding 7.8 g (22.8 mmol) of .beta.-D-lactose. Additional 5 ml of
the pH 8.5 buffer was added. The final white mixture was allowed to
stir for two days at room temperature. Fifty ml DI water were added
to the final dense white solution to ensure dissolution of all free
reagents. The final solution was filtered through a medium frit and
rinsed with 50 ml DI water. The wetness of the solid was
preserved.
Example 6
[0085] As an example of the experimental work, the synthesis of
lactose-[branching]-PGMA (FIG. 3-C-1), including a dendrimer,
followed these steps: Hundred mg of PGMA-NH.sub.2 (0.4 mmol
equivalents of the repeat unit) was mixed with 50 ml pH 8.5 20 mM
borate buffer and allowed to stir for few minutes at room
temperature. Sixty four mg of 1,1'-carbonyldiimidazole (0.4 mmol,
MW 162.15) was then added to the suspension and allowed to stir for
2 hours before adding 1 g of Hyperbranched bis-MPA
polyester-16-hydroxyl (0.57 mmol, 9.12 mmol.eq. OH). After two
additional hours, 1.48 g (9.12 mmol) of 1,1'-carbonyldiimidazole
was added to the suspension and allowed to stir for 2 more hours
before adding 15.6 g (45.6 mmol) of .beta.-D-lactose. The final
white mixture was allowed to stir for two days at room temperature.
Fifty ml DI water was added to the final dense white solution to
ensure dissolution of all free reagents. The final solution was
filtered through a medium frit and rinsed with 50 ml DI water. The
wetness of the solid was preserved.
Example 7
[0086] As an example of the experimental work, the synthesis of
lactose-[branching]-PGMA (FIG. 3-C-2), including chitosan, followed
these steps: Four hundred ml of 0.5% acetic acid in DI water was
prepared by adding 2 g of the acid to 400 mL of water. To this acid
solution, 2 g of Chitosan was added and the solution was allowed to
stir at room temperature for 5 min until becoming monophasic. Then,
200 mg of PGMA was added and the final suspension was allowed to
stir at room temperature for two hours. The final off-white
suspension was then filtered through a medium frit and the solid
was washed with 100 ml of DI water. The isolated solid was
re-suspended in 10 ml DI water. Its pH was ca. 4. One drop of a
sodium carbonate solution (5 wt. % sodium carbonate solution
prepared by dissolving 500 mg of Na.sub.2CO.sub.3 in 9.5 g DI
water) was added to increase the pH to ca. 9. The now basic mixture
was filtered and rinsed with 50 ml DI water. The yield was 140 mg
of chitosan-PGMA. Hundred mg of this intermediate was suspended in
10 ml pH 8.0 borate buffer. 0.148 g (0.9 mmol) of
1,1'-carbonyldiimidazole was added to the suspension and allowed to
stir for 2 hours before adding 1.56 g (4.5 mmol) of
.beta.-D-lactose. The final mixture was allowed to stir for two
days at room temperature. The final solution was filtered through a
medium frit, rinsed with 100 ml DI water.
Example 8
[0087] As yet another example of the experimental work, the
synthesis of lactose-[branching]-sand follows these steps: Five
grams of fine sand are vigorously stirred with 20 ml DI water, then
filtered through a medium frit. They are then mixed with 10 ml pH
8.5 20 mM borate buffer and allowed to stir for few minutes at room
temperature. Sixteen mg of 1,1'-carbonyldiimidazole (0.1 mmol, MW
162.15) are then added to the suspension and allowed to stir for 2
more hours before adding branched poly(ethylene glycol) (2.28
m-mmol.eq. OH). After two additional hours, 0.37 g (2.28 mmol) of
1,1'-carbonyldiimidazole is added to the suspension and allowed to
stir for 2 more hours before adding 3.9 g (11.4 mmol) of
.beta.-D-lactose. Five ml of the pH 8.5 borate buffer are then
added. The final mixture is allowed to stir for two days at room
temperature. The final solution is filtered through a medium frit
and rinsed with 50 ml DI water. The wetness of the solid is
preserved.
Example 9
[0088] As yet another example of the experimental work, the
synthesis of lactose-[branching]-Sepharose follows these steps: One
gram of wet Sepharose (ca. 5 wt. % in water) is mixed with 10 ml pH
8.5 20 mM borate buffer and allowed to stir for few minutes at room
temperature. Thirty two mg of 1,1'-carbonyldiimidazole (0.2 mmol,
MW 162.15) are then added to the suspension and allowed to stir for
2 more hours before adding branched poly(ethylene glycol) (4.56
mmol.eq. OH). After two additional hours, 0.74 g (4.56 mmol) of
1,1'-carbonyldiimidazole is added to the suspension and allowed to
stir for 2 hours before adding 7.8 g (22.8 mmol) of
.beta.-D-lactose. Additional 5 ml of the pH 8.5 buffer is added.
The final mixture is allowed to stir for two days at room
temperature. Fifty ml DI water are added to the final solution to
ensure dissolution of all free reagents. The final solution is
filtered through a medium frit and rinsed with 50 ml DI water. The
wetness of the solid is preserved.
Example 10
[0089] As yet another example of the experimental work, the
synthesis of lactose-[branching]-PGMA, including a branched
polymer, follows these steps: Hundred mg of PGMA-NH.sub.2 (0.4 mmol
equivalents of the repeat unit) are mixed with 50 ml pH 8.5 20 mM
borate buffer and allowed to stir for few minutes at room
temperature. Sixty four mg of 1,1'-carbonyldiimidazole (0.4 mmol,
MW 162.15) are then added to the suspension and allowed to stir for
2 hours before adding branched poly(ethylene glycol) (9.12 mmol.eq.
OH). After two additional hours, 1.48 g (9.12 mmol) of
1,1'-carbonyldiimidazole are added to the suspension and allowed to
stir for 2 more hours before adding 15.6 g (45.6 mmol) of
.beta.-D-lactose. The final mixture is allowed to stir for two days
at room temperature. Fifty ml DI water are added to the final
solution to ensure dissolution of all free reagents. The final
solution is filtered through a medium frit and rinsed with 50 ml DI
water. The wetness of the solid is preserved.
Example 11
[0090] As yet another example of the experimental work,
sialyllactose-complexed with PGMA was prepared. Since influenza's
envelope protein, hemagglutinin (HA), is known to strongly bind to
innate sialic acid in membranes of host cells, covalently attaching
sialyllactose onto insoluble supports would allow virus adsorption
to these surfaces. To this end, sialyllactose-complexed with PGMA
was prepared following FIG. 3-C-2 using 6'-sialyllactose instead of
.beta.-D-lactose as the starting material. The linker therein was
chitosan. Chemical derivatization of the material was monitored by
recombinant HA binding assays (quantified by the Bradford test)
(FIG. 9).
[0091] The PGMA-attached sialyllactose along with a set of controls
were tested in a buffered (PBS) aqueous solution of PR8 strain of
influenza-A virus, with the viral titers in the supernatants
quantified using the plaque assay. The results revealed that
PGMA-chitosan-lactose removed more than 98% of the virus from
solution (Table 1 and FIG. 10). Furthermore, data showed that the
virus adsorption to the disclosed material complexes follows a
linear isotherm; the relatively constant percentage of adsorbed
influenza A to the material complexes reflects Freundlich isotherm
that describes adsorption of entities on suspended surfaces at very
low surface coverage. Indeed, the linearity between log (adsorbed
virus) and log (initial virus) was confirmed by obtaining a R.sup.2
coefficient=0.994 (Table 2 and FIG. 11).
TABLE-US-00001 TABLE 1 Quantification of influenza A attachment to
insoluble materials Average # of virus in supernatant Standard
Captured [virus]% Material (.times.10{circumflex over ( )}3 pfu/ml)
Deviation compared to PBS PGMA 7.7 1.1 45 PGMA-Ch 8.7 2.3 38
PGMA-Ch-L 0.4 0.2 97 PGMA-Ch-SL 1.2 0.2 91 Ch 12.7 2.1 9 PBS, no 14
0.8 0 material PGMA = poly(glycidyl methacrylate), Ch = chitosan,
SL = sialyllactose, L = lactose
TABLE-US-00002 TABLE 2 Activity of complexed poly(glycidyl
methacrylate) polymer while varying the initial titer of influenza
A Starting [Virus] Adsorbed [Virus] (pfu/ml) (pfu/mg) % Adsorbed
[virus] 1,433,333 142133 99.2 28,333 2791 98.5 863 85 98.8 18000
1100 93.9
[0092] One skilled in the art will appreciate further features and
advantages of the invention based on the above-described
embodiments. Accordingly, the invention is not to be limited by
what has been particularly shown and described, except as indicated
by the appended claims. All publications and references cited
herein are expressly incorporated herein by reference in their
entirety.
* * * * *